A series of 2H- and 13C-labeled glutamates were used as substrates for coenzyme B12-dependent glutamate mutase, which equilibrates (S)-glutamate with (2S, 3S)-3-methylasparate. These compounds contained the isotopes at C-2, C-3 or C-4 of the carbon chain: [2-2H], [3-2H2], [4-2H2], [2,3,3,4,4-2H5], [2-13C], [3-13C], and [4-13C]glutamate. Each reaction was monitored by EPR spectroscopy and revealed a similar signal characterized by g'xy=2.1,g'z=1.985 and A'=5.0mT. The interpretation of the spectral data was aided by simulations which gave close agreement with experiment. This approach underpinned the idea of the formation of a radical pair, consisting of cob(II)alamin interacting with an organic radical at a distance of 6.6+/- 0.9A. Comparison of the hyperfine couplings observed with unlabeled glutamate with those from the labeled glutamates enabled a principle contributor to the radical pair to be identiied as the 4-glytamyl radical. These findings support the currently accepted mechanism for the glutamate mutase reaction, i.e. the process is initiated through hydrogen atom abstraction from C-4 of glutamate by the 5'-deoxyadenosyl radical, which is derived by homolysis of the Co-C sigma-bond o the coenzyme B21.